JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Protein kinase C regulates the expression of cell wall-related genes in RlmA-dependent and independent manners in Aspergillus nidulans.
Biosci. Biotechnol. Biochem.
PUBLISHED: 10-28-2014
Show Abstract
Hide Abstract
A protein kinase C of Aspergillus nidulans, PkcA, is required for cell wall integrity (CWI) and is considered a major component of the regulating pathway. To investigate whether PkcA regulates the transcription of cell wall-related genes, we constructed strains expressing pkcA(R429A) that encodes an activated form of PkcA. The mRNA levels of most chitin synthase genes and an ?-glucan synthase gene, agsB, were increased when pkcA(R429A) expression was induced. These mRNA increases were not observed or were only partially observed, in a deletion mutant of rlmA, an ortholog of RLM1 that encodes a transcription factor in the CWI pathway in Saccharomyces cerevisiae. In addition, in a pkcA temperature-sensitive mutant under heat stress, the mRNA levels of some chitin synthase genes and agsB did not increase. These results suggest that PkcA is involved in CWI maintenance through the transcriptional regulation of cell wall-related genes.
Related JoVE Video
Fatty Aldehyde Dehydrogenase Multigene Family Involved in the Assimilation of n-Alkanes in Yarrowia lipolytica.
J. Biol. Chem.
PUBLISHED: 10-16-2014
Show Abstract
Hide Abstract
In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (?hfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the ?hfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, while this elevation in FALDH activity by n-decane was not observed in the extract of the ?hfd1-4 strain. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, while Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that the Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.
Related JoVE Video
Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae ?pem1 ?pem2 double mutant without exogenous choline supply.
Biochim. Biophys. Acta
PUBLISHED: 05-01-2014
Show Abstract
Hide Abstract
In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3×HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1?pem2?) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1?pem2? strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.
Related JoVE Video
Human CTP:phosphoethanolamine cytidylyltransferase: enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-07-2014
Show Abstract
Hide Abstract
CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis-Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.
Related JoVE Video
Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-23-2014
Show Abstract
Hide Abstract
A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of (13)C-labeled diC8PC ((methyl-(13)C)3-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-(13)C)3-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.
Related JoVE Video
Identification and Characterization of a Gene Encoding an ABC Transporter Expressed in the Dicarboxylic Acid-Producing Yeast Candida maltosa.
Biosci. Biotechnol. Biochem.
PUBLISHED: 12-07-2013
Show Abstract
Hide Abstract
A gene, CmCDR1, encoding an ABC transporter of the dicarboxylic acid (DCA)-producing yeast Candida maltosa was cloned. Transcription of CmCDR1 was upregulated in a DCA-hyper-producing mutant of C. maltosa in a later phase of culture on n-dodecane, but not in its parental strain. CmCDR1 expression was significantly induced by the longer-chain DCA in this mutant.
Related JoVE Video
Phosphatidic acid and phosphoinositides facilitate liposome association of Yas3p and potentiate derepression of ARE1 (alkane-responsive element one)-mediated transcription control.
Fungal Genet. Biol.
PUBLISHED: 06-19-2013
Show Abstract
Hide Abstract
In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.
Related JoVE Video
Functional differentiation of chitin synthases in Yarrowia lipolytica.
Biosci. Biotechnol. Biochem.
PUBLISHED: 06-07-2013
Show Abstract
Hide Abstract
In this study, we identified seven chitin synthase-encoding genes in the genome of the dimorphic yeast Yarrowia lipolytica. Three encoded chitin synthases with myosin motor-like domains at their N-termini, and we designated these CSM1 to CSM3, whereas four were identified as CHS1 to CHS4. To investigate the functions of these seven genes, we constructed and characterized their deletion mutants. The chs2? mutant formed chained cells in which daughter cells were connected with mother cells and had abnormally thick septa at the bud neck. The chs4? mutant showed remarkably reduced chitin content in its cell wall. The chs2?, csm1?, and csm2? mutants were found to be highly sensitive to chitin binding dyes, calcofluor white (CFW) and Congo red, whereas the chs4? mutant was resistant to CFW. These results suggest that Chs2 and Chs4 play major roles in septum formation and cell wall chitin synthesis respectively, whereas Csm1 and Csm2 are involved in the maintenance of cell wall architecture and/or cell wall integrity. The populations of filamentous cells, a type of cell population that are defined by the lengths of the cellular long and short axes, decreased in the chs3? mutant, suggesting that Chs3 is involved in cellular morphogenesis.
Related JoVE Video
Myosin motor-like domain of class VI chitin synthase CsmB of Aspergillus nidulans is not functionally equivalent to that of class V chitin synthase CsmA.
Biosci. Biotechnol. Biochem.
PUBLISHED: 02-07-2013
Show Abstract
Hide Abstract
Chitin is a major cell wall component of many filamentous fungi. Among the eight chitin synthase genes of Aspergillus nidulans, csmA and csmB encode a myosin motor-like domain (MMD) and a chitin synthase domain (CSD) at their N- and C-termini respectively. In our previous reports, we suggested that CsmA and CsmB play compensatory roles essential for polarized hyphal growth although their functions do not completely overlap, and that their MMDs are essential for their functions. In the present study, we constructed chimeric csm genes by swapping N-terminal MMD-encoding halves of csmA and csmB and studied them to identify functional differences in the MMDs. Expression of the chimeric gene encoding the MMD-including half of CsmA (MA) and the CSD-including half of CsmB thoroughly suppressed the phenotypic defects of the ?csmB mutant, whereas the chimeric gene encoding the MMD-including half of CsmB (MB) and the CSD-including half of CsmA did not fully suppress the defects of the ?csmA mutant, suggesting that MA suffices for the function of MB while MB is not functionally equivalent to MA.
Related JoVE Video
Transcriptional repression by glycerol of genes involved in the assimilation of n-alkanes and fatty acids in yeast Yarrowia lipolytica.
FEMS Yeast Res.
PUBLISHED: 01-14-2013
Show Abstract
Hide Abstract
The yeast Yarrowia lipolytica assimilates n-alkanes or fatty acids as carbon sources. Transcriptional activation by n-alkanes of ALK1 encoding a cytochrome P450 responsible for the terminal hydroxylation has been well studied so far, but its regulation by other carbon sources is poorly understood. Here, we analyzed the transcriptional regulation of ALK1 by glycerol. Glycerol is a preferable carbon source compared to glucose for Y. lipolytica. The n-decane-induced transcript levels of ALK1 as well as the reporter gene under the control of ALK1 promoter were significantly decreased in the simultaneous presence of glycerol, but not of glucose. Similarly, the expression of PAT1 encoding acetoacetyl-CoA thiolase involved in ?-oxidation was induced by n-decane or oleic acid, but its transcript level was decreased when glycerol was supplemented. These results indicate that glycerol represses the transcription of the genes involved in the metabolism of hydrophobic carbon sources in Y. lipolytica. Repression of ALK1 transcription by glycerol was not observed in the deletion mutant of GUT1 encoding glycerol kinase, implying that the phosphorylation of glycerol is required for the glycerol repression.
Related JoVE Video
Involvement of Golgi-associated retrograde protein complex in the recycling of the putative Dnf aminophospholipid flippases in yeast.
Biochem. Biophys. Res. Commun.
PUBLISHED: 11-24-2011
Show Abstract
Hide Abstract
It is widely accepted that phosphatidylethanolamine (PE) is enriched in the cytosolic leaflet of the eukaryotic plasma membranes. To identify genes involved in the establishment and regulation of the asymmetric distribution of PE on the plasma membrane, we screened the deletion strain collection of the yeast Saccharomyces cerevisiae for hypersensitive mutants to the lantibiotic peptide Ro09-0198 (Ro) that specifically binds to PE on the cell surface and inhibits cellular growth. Deletion mutants of VPS51, VPS52, VPS53, and VPS54 encoding the components of Golgi-associated retrograde protein (GARP) complex, YPT6 encoding a Rab family small GTPase that functions with GARP complex, RIC1 and RGP1 encoding its guanine nucleotide exchange factor (GEF), and TLG2 encoding t-SNARE exhibited hypersensitivity to Ro. The mutants deleted for VPS51, VPS52, VPS53, and VPS54 were impaired in the uptake of fluorescently labeled PE. In addition, aberrant intracellular localization of the EGFP-tagged Dnf2p, the putative inward-directed phospholipid translocase (flippase) of the plasma membrane, was observed in the mutant defective in the GARP complex, Ypt6p, its GEF proteins, or Tlg2p. Our results suggest that the GARP complex is involved in the recycling of Dnf flippases.
Related JoVE Video
Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7.
DNA Res.
PUBLISHED: 09-06-2011
Show Abstract
Hide Abstract
The term sake yeast is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.
Related JoVE Video
Construction and characterization of a Yarrowia lipolytica mutant lacking genes encoding cytochromes P450 subfamily 52.
Fungal Genet. Biol.
PUBLISHED: 07-25-2011
Show Abstract
Hide Abstract
The initial hydroxylation of n-alkane is catalyzed by cytochrome P450ALK of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica. A mutant with a deletion of all 12 genes, ALK1 to ALK12, which are deduced to encode cytochromes P450 of the CYP52 family in Y. lipolytica, was successfully constructed. This deletion mutant, ?alk1-12, completely lost the ability to grow on n-alkanes of 10-16 carbons. In contrast, ?alk1-12 grew on the metabolite of n-dodecane, i.e., n-dodecanol, n-dodecanal, or n-dodecanoic acid, as well as the wild-type strain. In addition, production of n-dodecanoic acid was not observed when ?alk1-12 was incubated in the presence of n-dodecane. These results indicate the essential roles of P450ALKs in the oxidation of n-alkane. ?alk1-12 will be valuable as a host strain to express an individual ALK gene to elucidate the molecular function and substrate specificity of each P450ALK. Transcriptional activation of the ALK1 promoter by n-alkanes was observed in ?alk1-12 as in the wild-type strain, implying that n-alkanes per se, but not their metabolites, trigger n-alkane-induced transcriptional activation in Y. lipolytica.
Related JoVE Video
Construction of high sensitive detection system for endocrine disruptors with yeast n-alkane-assimilating Yarrowia lipolytica.
J. Microbiol. Biotechnol.
PUBLISHED: 12-03-2010
Show Abstract
Hide Abstract
To construct a highly sensitive detection system for endocrine disruptors, we have compared the activity of promoters with the ALK1, ICL1, RPS7 and TEF1 for heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of lacZ or hERalpha reporter gene, respectively, and the activity was evaluated by beta-galactosidase assay by lacZ or western blot analysis by hERalpha. The expression analysis revealed that the ALK1 and ICL1 promoter were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly high level of expression in the presence of glucose and glycerol, respectively. Particularly, the TEF1 promoter showed the highest beta-galactosidase activity and a significant signal by western blotting with the anti-estrogen receptor compared with the other promoters. Moreover, the detection system was constructed with promoters were linked to the upstream of expression vector for hERalpha gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERalpha and CXAU1-2XERE was the most effective system for the E2-dependent induction of the beta-galactosidase activity. This system showed the highest beta-galactosidase activity at 10-6 M E2 and the activity could be detected at even the concentration of 10-10 M E2. As the result, we constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity and reproducibility of the system for characterizing environmental estrogens.
Related JoVE Video
An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica.
Biochem. Biophys. Res. Commun.
PUBLISHED: 10-13-2010
Show Abstract
Hide Abstract
The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in ?-oxidation and peroxisome proliferation by oleate was distinctly diminished in the ?por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.
Related JoVE Video
Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast.
Biochim. Biophys. Acta
PUBLISHED: 01-06-2010
Show Abstract
Hide Abstract
Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn-2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn-2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.
Related JoVE Video
Myosin motor-like domain of the class VI chitin synthase CsmB is essential to its functions in Aspergillus nidulans.
Biosci. Biotechnol. Biochem.
PUBLISHED: 05-07-2009
Show Abstract
Hide Abstract
Chitin is one of the major cell wall components of ascomycete filamentous fungi, and chitin synthesis plays important roles in the morphogenesis of hyphae. In the Aspergillus nidulans genome, there are two genes, csmA and csmB, that encode a myosin motor-like domain (MMD) at their N-termini and a chitin synthase domain (CSD) at their C-termini. In our previous studies, we found that the MMD of CsmA was required for its functionality, and that CsmA and CsmB had certain overlapping functions essential for polarized filamentous growth. In this study, we constructed a strain that produced CsmB lacking the MMD (CSDeltaMB). This strain exhibited defects similar to those of the csmB deletion mutant. FLAG-tagged CSDeltaMB (CSDeltaMB-FLAG) did not properly localize to the hyphae. CsmB was co-immunoprecipitated with actin in vivo, whereas CSDeltaMB was not. These results suggest that the MMD of CsmB is crucial for its proper localization via interaction with actin.
Related JoVE Video
Class III chitin synthase ChsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development.
Eukaryotic Cell
PUBLISHED: 05-01-2009
Show Abstract
Hide Abstract
Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.
Related JoVE Video
Involvement of LEM3/ROS3 in the uptake of phosphatidylcholine with short acyl chains in Saccharomyces cerevisiae.
Biosci. Biotechnol. Biochem.
PUBLISHED: 03-07-2009
Show Abstract
Hide Abstract
A Saccharomyces cerevisiae strain deficient in phosphatidylethanolamine methyltransferase that exhibits choline auxotrophy grew in the presence of dioctanoyl phosphatidylcholine (diC8PC). We isolated and characterized mutants defective in growth in the diC8PC-containing medium. Mutations in LEM3/ROS3 impaired growth in that medium, indicating that Lem3p is involved in the utilization of extracellular phosphatidylcholine (PC) with short acyl chains.
Related JoVE Video
Yas3p, an Opi1 family transcription factor, regulates cytochrome P450 expression in response to n-alkanes in Yarrowia lipolytica.
J. Biol. Chem.
PUBLISHED: 01-08-2009
Show Abstract
Hide Abstract
In the alkane-assimilating yeast Yarrowia lipolytica, the expression of ALK1, a gene encoding cytochrome P450 that catalyzes the first step of n-alkane oxidation, is induced by n-alkanes. We previously demonstrated that two basic helix-loop-helix proteins, Yas1p and Yas2p, activate the transcription of ALK1 in an alkane-dependent manner by forming a heterocomplex and binding to alkane-responsive element 1 (ARE1), a cis-acting element in the ALK1 promoter. Here we identified an Opi1 family transcription factor, Yas3p, involved in the alkane-dependent transcription regulation of ALK genes. Deletion of YAS3 caused a significant increase in ALK1 mRNA in cells grown on glucose, glycerol, and n-alkanes. The YAS3 deletion also resulted in a marked elevation of reporter gene expression driven by an ARE1-containing promoter on glycerol and n-decane. Bacterially expressed Yas3p bound specifically to Yas2p, but not to Yas1p, in vitro. In addition, although green fluorescent protein-tagged Yas3p was localized in the nucleus in glucose-containing medium, it changed its localization to an endoplasmic reticulum-like compartment upon transfer to medium containing n-decane. These findings suggest that Yas3p functions as a master regulator of transcriptional response, which changes its localization between the nucleus and endoplasmic reticulum membrane in response to different carbon sources. Furthermore, quantitative real time PCR analysis of 12 ALK genes in YAS1, YAS2, and YAS3 deletion mutants suggested that Yas3p is involved in the transcriptional repression of a variety of ALK genes, including ALK1. In contrast, YAS3 deletion did not affect the mRNA level of an INO1 ortholog in Y. lipolytica, indicating functional diversity of Opi1 family transcription factors.
Related JoVE Video
Involvement of protein kinase C in the suppression of apoptosis and in polarity establishment in Aspergillus nidulans under conditions of heat stress.
PLoS ONE
Show Abstract
Hide Abstract
The pkcA gene, which encodes a protein kinase C (PKC) in the filamentous fungus Aspergillus nidulans, is essential for its viability. However, little is known about its functions. To address this issue, we constructed and characterized temperature-sensitive mutants of pkcA. The conidia of these mutants swelled slightly and exhibited apoptotic phenotypes at 42°C. The apoptotic phenotypes were suppressed by an osmotic stabilizer. Under these conditions, the conidia swelled extensively and did not form germ tubes. Moreover, polarized distribution of F-actin was not observed. We then utilized deletion mutants of bckA, an ortholog of Saccharomyces cerevisiae bck1 that encodes a mitogen-activated protein (MAP) kinase kinase kinase and functions downstream of PKC in the cell wall integrity pathway. These mutants exhibited apoptotic phenotypes at 42°C, but they did not show defects in polarity establishment under osmotically stabilized conditions. These results suggest that PkcA plays multiple roles during germination under conditions of heat stress. The first of these roles is the suppression of apoptosis induction, while the other involves polarity establishment. The former depends on the MAP kinase cascade, whereas the latter does not. In addition, repolarization, which was observed after depolarization in the wild-type strain and the bckA deletion mutant under conditions of heat stress, was not observed in the pkcA-ts mutant. This suggests that PkcA also plays role in polarity establishment during hyphal growth independent of the MAP kinase cascade under these conditions.
Related JoVE Video
Organotin compounds act as inhibitor of transcriptional activation with human estrogen receptor.
J. Microbiol. Biotechnol.
Show Abstract
Hide Abstract
In aquatic invertebrates, particularly marine gastropods, organotin compounds induce irreversible sexual abnormality in females, which is termed imposex, at very low concentrations. Organotin compounds are agonists for nuclear receptors such as RXRs and PPARgamma. However, the imposex phenomenon has not been reported to act as an antagonist on estrogen receptors in other species, including vertebrates and invertebrates. In order to gain insights into the antagonistic activity of organotin compounds on estrogen receptors (ERs), we examined the inhibitive effect of these compounds on estradiol-dependent beta-galactosidase activity using the yeast two-hybrid detection system consisting of a combination of the human estrogen receptor (hERbeta) ligand-binding domain and the co-activator steroid receptor co-activator-1 (SRC1). Tributyltin-hydroxide (TBT-OH) and triphenyltin-chlorine (TPT-Cl) exhibited an inhibitive effect on E?-dependent transcriptional activity, similar to antagonistic chemicals such as 4-hydroxytamoxifen (OHT) or ICI 182,780, at a very low concentration of 10?¹? M TBT or 10?¹? M TPT, respectively. The yeast growth and transcriptional activity with transcriptional factor GAL4 did not exhibit any effect at the tested concentration of TBT or TPT. Moreover, the yeast two-hybrid system using the interaction between p53 and the T antigen of SV40 large did not describe any effect at the tested concentration of OHT or ICI 182,780. However, the interaction between p53 and T antigen was inhibited at a TBT or TPT concentration of 10?? M, respectively. These results indicate that TBT and TPT act as inhibitors of ER-dependent reporter gene transcriptional activation and of the interaction between hERbeta LBD and the co-activator SRC1 in the yeast two-hybrid system. Consequently, our data could partly explain the occurrence of organotin compound-induced imposex on the endocrine system of mammals, including humans.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.